The long-range objectives of the research proposed in this application are to characterize the ligation structures of nickel in two important classes of nickel enzymes and to further develop the pulsed-EPR techniques of electron spin-echo envelope modulation (ESEEM) and electron spin-echo detected ENDOR spectroscopy for the characterization of paramagnetic centers in biological systems. The specific experiments aimed at achieving the first goal involve the study of Ni(III) and Ni(I) model complexes of biologically relevant structure using cw-EPR, cw-ENDOR, ESEEM, and ESE- ENDOR spectroscopies. Ni(III) peptide model complexes will be studied using ESEEM, cw- and ESE-ENDOR methods to obtain magnetic coupling parameters and coupling tensor orientations for 14N, 2H, and 1H containing ligands to determine how these magnetic parameters are related to structure. These data will provide the necessary background for interpreting the results of published and ongoing ESE and ENDOR investigations of the nickel center in Ni-Fe hydrogenases. Ni(I) derivatives of cofactor F430, thought to play a key role in methane production by methanogenic bacteria, will also be studied using pulsed- and cw-EPR techniques. Here, ligand hyperfine coupling constants and tensor orientations will yield detailed information concerning the axial bonding properties of the cofactor and the consequences of cofactor reduction as related to changes in corphin macrocycle distortion and "puckering". These properties are thought to be key elements in defining the unique chemistry of F430 and its catalytic function in methyl reductases. During the course of this research, ESE-ENDOR methodology will be developed and its capabilities compared directly with those of cw-ENDOR. In addition, current methods of data analysis will be improved. Because metalloenzymes serve as catalysts for a number of essential oxidation-reduction reactions in biological systems with paramagnetic ions being involved at some stage in these processes, the results of this research will provide pertinent information and enhanced spectroscopic tools for the investigation of many health-related problems.